Migraine is a painful and debilitating neurological condition involving the neuropeptide calcitonin gene-related peptide (CGRP). It affects 15% of the population and is the second leading cause of years lived with a disability. Indeed, over 40% of women will experience migraine during their lifetime. An exciting development is the recent FDA approval of a new class of CGRP-targeted drugs designed to prevent migraine. These drugs block CGRP or its canonical receptor. While effective for many people, a short-coming is that these drugs meet a clinically relevant endpoint for only about half the patients. To improve the efficacy of CGRP-based drugs and pave the way for new drug development, we need to know more about how CGRP works in migraine. We propose that a second CGRP receptor holds the key to developing new and improved migraine medications. This novel, high affinity CGRP receptor, AMY1, was first identified as a receptor for the related peptide amylin. A major barrier is a lack of fundamental knowledge about which of the two CGRP receptors contribute to specific aspects of migraine. The objective of the proposed research is to identify the specific CGRP receptor(s) that trigger migraine-like symptoms. Migraine involves increased sensitivity to CGRP, as supported by clinical studies showing that CGRP is both required and sufficient to cause migraine. However, in contrast to the peptide ligand, the identity of the CGRP receptor in migraine is open to debate. We have shown that two high affinity CGRP receptors exist. A critical need is to now determine which of the CGRP receptors act in defined regions to promote pain/aversion behaviors. We hypothesize that AMY1 is a novel and unexplored target that mediates specific behaviors of CGRP in the brain and/or periphery to cause migraine. We propose two aims that use unique experimental approaches to validate AMY1 as a novel target in the treatment of migraine. In Aim 1, we will comprehensively identify the location of each CGRP receptor in central and peripheral sites of the pain matrix. In Aim 2, we will determine whether one or both CGRP receptors contribute to migraine-like symptoms at various sites. Complementary genetic and pharmacological strategies will be used in the two aims. In particular, the use of a temporally controlled recombinase strategy for intersectional labeling will provide an innovative approach for validating the identity and role(s) of cells that co- express both subunits of either the AMY1 or canonical CGRP receptors. The outcome of these studies will be robust characterization of CGRP receptor involvement in migraine, which may challenge the current dogma that only one CGRP receptor is relevant in migraine. Validation of specific CGRP receptor involvement will be pivotal for rational drug design efforts and create a new direction for the development of migraine drugs that specifically target the CGRP and AMY1 receptors. Importantly, the development of novel CGRP-directed therapeutics will provide alternatives to opioids for management of migraine and potentially other chronic pain conditions.